Roles of G(o)alpha tryptophans in GTP hydrolysis, GDP release, and fluorescence signals.
Single tryptophan mutants of a histidine-tagged G(o)alpha (W132F and W212F) were prepared to examine the functional and spectroscopic role of tryptophan in G(o)alpha. The mutants bound GTP gamma S with high affinity and showed only modest changes in GDP affinity. GTP gamma S-stimulated intrinsic fluorescence changes were completely abolished by removal of W212 but were not affected by elimination of W132. In contrast, both W132 and W212 contributed to the fluorescence signal from binding of methylanthraniloyl-GTP gamma S (mGTP gamma S). W132F and W212F mutants showed 57% and 34% of the mGTP gamma S fluorescence change of wild type (WT), respectively. The decreased fluorescence signals were not due to reduced activation of the W212F protein by nucleotide as protection from tryptic digestion was unchanged. The kinetics of nucleotide binding and hydrolysis were also altered in both mutants. GDP dissociation was slower (0.14 min-1) for W132F and faster (0.54 min-1) for W212F than for WT (0.25 min-1). As expected, the steady-state Vmax for GTPase was lower for W132F, but surprisingly it was also lower for W212F despite faster GDP release. Single turnover kinetics revealed a lower kcat for W212F (0.52 min-1) compared to WT (1.39 min-1) and W132F (1.0 min-1). Thus, W212 in G(o)alpha makes a dominant contribution to both intrinsic and extrinsic fluorescence signals upon alpha subunit activation. In addition, both tryptophans modulate the kinetics of nucleotide binding and hydrolysis.
Lan
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Department of Pharmacology, University of Michigan, Ann Arbor 48109-0632, USA.
Remmers
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Neubig
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D019943:Amino Acid Substitution / Q000235:genetics; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics; D062367:ortho-Aminobenzoates
D062367:ortho-Aminobenzoates; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010649:Phenylalanine; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go